Early Spermiogenesis Research Articles

The sperm ultrastructure of Spermophagus kuesteri Schilsky, 1905 (Chrysomelidae, Bruchinae) and considerations on the relationships of Cucujiformia superfamilies

The sperm ultrastructure of the bean-weevil Spermophagus kuesteri (Bruchinae) was studied to verify the congruence of the new position of the subfamily within Chrysomelidae. The results indicated a positive answer to the question supporting a close relationship between Chrysomelidae and Curculionidae, a finding confirmed also by molecular data. Moreover, the sperm morphology of Divales cinctus, a member of Melyridae (Cleroidea) allowed to confirm the different sperm organization between members of this superfamily and Phytophaga (Chrysomeloidea + Curculionoidea). While studying the spermiogenesis of S. kuesteri, some sperm cysts showed aberrant cells provided with two flagella in the same plasma membrane. These aberrant sperm could be the result, during early spermiogenesis, of irregular processes involving the canal rings between spermatids.

Dynamic rearrangement and autophagic degradation of mitochondria during spermiogenesis in the liverwort Marchantia polymorpha.

Mitochondria change their morphology in response to developmental and environmental cues. During sexual reproduction, bryophytes produce spermatozoids with two mitochondria in the cell body. Although intensive morphological analyses have been conducted, how this fixed number of mitochondria is realized remains poorly understood. Here, we investigate how mitochondria are reorganized during spermiogenesis in Marchantia polymorpha. We find that the mitochondrial number is reduced to one through fission followed by autophagic degradation during early spermiogenesis, and then the posterior mitochondrion arises by fission of the anterior mitochondrion. Autophagy is also responsible for the removal of other organelles, including peroxisomes, but these other organelles are removed at distinct developmental stages from mitochondrial degradation. We also find that spermiogenesis involves nonautophagic organelle degradation. Our findings highlight the dynamic reorganization of mitochondria, which is regulated distinctly from that of other organelles, and multiple degradation mechanisms operate in organelle remodeling during spermiogenesis in M.polymorpha.

Bryophyte Spermiogenesis Occurs Through Multimode Autophagic and Nonautophagic Degradation

Mitochondria change their morphology in response to developmental and environmental cues. During sexual reproduction, bryophytes produce spermatozoids with two mitochondria in the cell body. Although intensive morphological analyses have been conducted thus far, how this fixed number of mitochondria is realized remains unknown. Here, we investigated how mitochondria are reorganized during spermiogenesis in Marchantia polymorpha. We found that the mitochondrial number is reduced to one through fission followed by autophagic degradation during early spermiogenesis, and then the posterior mitochondrion arises by fission of the anterior mitochondrion. Autophagy is also responsible for the removal of other organelles, including peroxisomes, but these other organelles are removed at distinct developmental stages from mitochondrial degradation. We also found that spermiogenesis involves nonautophagic organelle degradation. Our findings highlight the dynamic reorganization of mitochondria, which is regulated distinctly from that of other organelles, and multiple degradation mechanisms operate in organelle remodeling during spermiogenesis in M. polymorpha.

Inhibition of autophagy impairs acrosome and mitochondrial crista formation during spermiogenesis in turtle: Ultrastructural evidence

Autophagy is a subcellular process that is extensively involved in spermiogenesis. In this study, we observed ultrastructural malformation of acrosome and mitochondrial cristae during the spermiogenesis of Chinese soft-shelled turtle due to the inhibition of autophagy. Autophagy was blocked with 3-MA, and the inhibition of autophagy was confirmed through western blot analysis. The morphological abnormalities of acrosomes and mitochondria were observed under transmission electron microscopy (TEM). In the early spermiogenesis (Golgi and cap phases), damaged macrovesicle was observed, and its proper expansion over the nucleus failed to be form a normal acrosomal cap. As spermiogenesis proceeded, the malformation of the acrosome in spermatids became more severe. In the late spermiogenesis (acrosomal and maturation phases), defective acrosome with damaged acrosomal membrane that was detached from the nucleus was observed. Along with malformed acrosome, elongation failed nucleus having oval or round shaped morphology was also observed. Moreover, morphological damage to the mitochondrial cristae was observed. Lacuna formation, half and complete loss of cristae were observed in the mitochondria of developing spermatids. We proposed that autophagy is required for normal formation of the acrosome and mitochondrial cristae during turtle spermiogenesis.

Brg1 subunit of chromatin remodelling complex is present during Chara vulgaris (Charophyceae) spermatogenesis

ABSTRACTDuring spermatogenesis, cells developed as a result of numerous mitotic and meiotic divisions transform into mature spermatozoids. In spermatids, remodelling of chromatin structure takes place which is connected with nuclear protein exchange, DNA double strand breaks and epigenetic modifications. Chromatin remodelling complexes, which have mostly been studied in animals, also participate in this process. The Brg1 protein, a functional homologue of the yeast Swi2/Snf2 catalytic subunit of the SWI/SNF complex, is engaged in regulation of cell proliferation and highly expressed in round spermatids in mammals. Immunocytochemical studies with the anti-Brg1 antibody revealed positive reactions in nuclei of the green alga Chara vulgaris at the 64-cell proliferative stage and in spermatid nuclei at the I/II–VII spermiogenesis stages. The most intensive reaction was observed at the early spermiogenesis stages (I/II–III), while at the initial stages of a proliferative phase (4-, 8- and 16-cell) the reaction was not observed, and at 32-cell and VII stages the immunosignals were very weak. Ultrastructural studies with the immunogold technique confirmed the results of the immunocytochemical studies. The highest numbers of gold grains were observed at stages I/II and III of spermiogenesis, and together they constituted above 48% of the total number of gold grains. A much lower, but still substantial, amount of these grains was observed at the 64-cell stage and IV stage (>15% and 17%), respectively. Percentage analysis revealed the lowest number of gold particles at stage VII (3.72%). The significant presence of Brg1 protein at early spermiogenesis stages is correlated with acetylation of the H4K12 histone. It may also be hypothesized that in C. vulgaris the Brg1 subunit participates in processes important for proper chromatin condensation and facilitates maintenance of the correct shape of the spermatid nucleus. On the basis of earlier and current studies it seems that chromatin remodelling in spermatids of this model alga proceeds according to the model presented for mammals.

Ultrastructural investigations on spermatogenesis and spermatozoan morphology in the endangered Adriatic sturgeon, Acipenser naccarii (Chondrostei, Acipenseriformes).

Spermatogenesis was investigated in the Adriatic sturgeon, Acipenser naccarii, by light and electron microscopy. The testis of the unrestricted type had a germinal compartment composed of lobules containing germ cells and Sertoli cells, and separated by a basal lamina from the interstitial compartment, in which Leydig and myoid cells were detected for the first time in Acipenseridae. Spermatogenesis occurred in spermatocysts produced when Sertoli cells became associated with type A spermatogonia of subsequent generations, which produced a clone of synchronized aligned spermatogonia. In primary spermatocytes at zygo-pachytene stage, the large spherical nucleus contained synaptonemal complexes. The smaller secondary spermatocytes were ovoid with a central round nucleus and scarce cytoplasm. Spermatids were interconnected by cytoplasmic bridges until early spermiogenesis. Chromatin initially condensed as long, twisted, and nonhomogeneous fibers and finally as a compact structure made of thick filaments. Early spermatids showed the flagellum, the primordia of centriole complex and of "implantation fossa," followed by the acrosomal vesicle formed by Golgi complexes and a fibrous body associated to centriole complex. The spermatozoan head had 10 postero-lateral projections and a trapezoidal nucleus, a cylindrical midpiece with six to eight mitochondria, the centriole complex, and a "9 + 2" flagellum with a pair of lateral fins. Three helical endonuclear canals crossed the nucleus from the acrosome base to the implantation fossa; their spiralization and that of chromatin fibers suggest a spiral twisting of the nucleus during spermiogenesis. The Sertoli cells performed phagocytosis of degenerating spermatids and spermatozoa. Significant interindividual differences were detected in most morphological parameters of spermatozoa. Data on spermatogenesis in A. naccarii and morphometric measurements on mature spermatozoa provide information about the reproductive biology of the species useful not only for phylogenetic studies but also for evaluation of sperm quality for artificial reproduction projects and restocking of this and other critically endangered sturgeon species.

Ultrastructure of spermiogenesis and spermatozoa in Marchalina hellenica (Gennadius) (Hemiptera: Sternorrhyncha, Marchalinidae)

The spermiogenesis, the sperm structure and the sperm motility of Marchalina hellenica (Gennadius) were examined. In the early spermiogenesis a centriolar apparatus was identified, but this structure is not involved in the production of the sperm flagellum. As in other Coccoidea, the flagellar axoneme originates by the activity of the thickened tip of the numerous microtubules surrounding the nuclear anterior region close to the periphery of the cell. This region pushes against a narrow cytoplasmic layer, giving rise to a papilla. In this region a novel structure, consisting of a regular network of thin filaments, arranged orthogonally to the bundle of microtubules, is visible. The sperm flagellum consists of a series of about 260 microtubules, regularly arranged in rings around the axial nucleus. This latter extends in the middle part of the sperm length. As usual in scale insects, sperm form a bundle, which in M. hellenica is composed of 64 sperm cells, surrounded by somatic cyst cells. The sperm bundle has an helicoidal array, with a cap of dense material at its apex, lending the anterior and the posterior region of the sperm bundle with a different structural organization. This difference is responsible of the different speed gradient observed in the helical wave propagating along the sperm bundle.

Atypical centrioles are present in Tribolium sperm.

Typical centrioles are made of microtubules organized in ninefold symmetry. Most animal somatic cells have two centrioles for normal cell division and function. These centrioles originate from the zygote, but because the oocyte does not provide any centrioles, it is surprising that the zygotes of many animals are thought to inherit only one centriole from the sperm. Recently, in the sperm of Drosophila melanogaster, we discovered a second centriolar structure, the proximal centriole-like structure (PCL), which functions in the zygote. Whether the sperm of other insects has a second centriolar structure is unknown. Here, we characterized spermiogenesis in the red flour beetle, Tribolium castaneum. Electron microscopy suggests that Tribolium has one microtubule-based centriole at the tip of the axoneme and a structure similar to the PCL, which lacks microtubules and lies in a cytoplasmic invagination of the nucleus. Immunostaining against the orthologue of the centriole/PCL protein, Ana1, also recognizes two centrioles near the nucleus during spermiogenesis: one that is microtubule-based at the tip of the axoneme, suggesting it is the centriole; and another that is more proximal and appears during early spermiogenesis, suggesting it is the PCL. Together, these findings suggest that Tribolium sperm has one microtubule-based centriole and one microtubule-lacking centriole.

Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse

Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis.

Ultrastructure of spermatogenesis and mature spermatozoa in the flatworm Prosthiostomum siphunculus (Polycladida, Cotylea).

This is the first study investigating spermatogenesis and spermatozoan ultrastructure in the polyclad flatworm Prosthiostomum siphunculus. The testes are numerous and scattered as follicles ventrally between the digestive ramifications. Each follicle contains the different stages of sperm differentiation. Spermatocytes and spermatids derive from a spermatogonium and the spermatids remain connected by intercellular bridges. Chromatoid bodies are present in the cytoplasm of spermatogonia up to spermatids. During early spermiogenesis, a differentiation zone appears in the distal part of spermatids. A ring of microtubules extends along the entire sperm shaft just beneath the cell membrane. An intercentriolar body is present and gives rise to two axonemes, each with a 9 + "1" micro-tubular pattern. Development of the spermatid leads to cell elongation and formation of a filiform, mature spermatozoon with two free flagella and with cortical microtubules along the sperm shaft. The flagella exit the sperm shaft at different levels, a finding common for acotyleans, but so far unique for cotylean polyclads. The Golgi complex produces numerous electron-dense bodies of two types and of different sizes. These bodies are located around a perinuclear row of mitochondria. The elongated nucleus extends almost along the entire sperm body. The nucleus is wide in the proximal part and becomes narrow going towards the distal end. Thread-like chromatin mixed with electron-dense intranuclear spindle-shaped bodies are present throughout nucleus. The general sperm ultrastructure, the presence of intranuclear bodies and a second type of cytoplasmic electron-dense bodies may provide characters useful for phylogenetic analysis.

Giant spermatozoa of Diasemopsis (Diopsidae, Diptera) – Structural, ultrastructural and functional aspects

The spermatozoa of Diasemopsis comoroensis and Diasemopsis meigenii differ from the conventional brachyceran type in several respects. Not only are they very long but they are also extraordinarily wide, especially at the very end of the tail. The latter is effected by two cellular components: oversized mitochondrial derivatives and a prominent central band, which is a peculiar structure not known from any other spermatozoa. Based on its position with respect to the other organelles and its origin during early spermiogenesis, the central band is interpreted as a derivative of the centriolar adjunct material. Like the axoneme, the mitochondrial derivatives and the central band extend through the entire length of the spermatozoon tail. The spermatozoon tail is helical and can be coiled up into a tight cone-shaped spiral with a peculiar corkscrew shaped end portion. The potential adaptive significance of these features and their coevolution with the morphology of the female's multi-chambered ventral receptacle are discussed, as are sperm competition and cryptic female choice.

Expression of cubilin in mouse testes and Leydig cells.

Cubilin (cubn) is a receptor for vitamins and various protein ligands. Cubn lacks a transmembrane domain but anchors to apical membranes by forming complexes with Amnionless or Megalin. In an effort to better understand the uptake of nutrients in testis, we analysed cubn expression in the developing mice testes. In testes, cubn mRNA increased from birth to adulthood. In the inter-stitium and isolated seminiferous tubules, neonatal increase in cubn mRNA until 14 days post-partum (pp) was followed by a marked increase at puberty (28 days pp). Cubn was found in the gonocytes, spermatogonia, spermatocytes and spermatids in the developing testes. In adult testes, strong Cubn immunoreactivity was found in the elongating spermatids, suggesting the role of Cubn in endocytosis during early spermiogenesis. In Sertoli cells and peritubular cells, Cubn immunoreactivity was weak throughout the testis development. In the inter-stitium, Cubn immunoreactivity was found in foetal Leydig cells, was weak to negligible in the stem cells and progenitor Leydig cells and was strong in immature and adult Leydig cells, demonstrating a positive association between Cubn and steroidogenic activity of Leydig cells. Collectively, these results suggest that Cubn may participate in the endocytotic uptake of nutrients in germ cells and somatic cells, supporting the spermatogenesis and steroidogenesis in mouse testes.

Ultrastructural observations on spermiogenesis in the peanut worm, Phascolosoma esculenta (Sipuncula: Phascolosomatidea)

Ultrastructural characteristics of spermiogenesis in the peanut worm, Phascolosoma esculenta (Phascolosomatidea), were observed by transmission electron microscopy (TEM). The spermiogenesis principally occurs in the coelom and can be divided into three stages (the early, middle and late stages) based on the chromatin morphology, acrosome development, spermatozoon midpiece and flagellum formation. Spermatids within a given spermatid mass develop synchronously. With the spermiogenesis proceeding, the spermatid masses become loosely structured, and later, adjacent spermatids are interconnected at one extremity of the cells. Gradually, condensation of the chromatin accelerates and is almost completed in late spermiogenesis, leaving the late spermatids with highly condensed homogeneous chromatin. In the spermatid head, the conical acrosome is generally composed of an acrosomal vesicle which is formed by the coalescence of small proacrosomal vesicles within the cytoplasm, a subacrosomal space that situates between the acrosomal vesicle and nucleus, and an acrosomal rod which develops from a bunch of filamentous material within the subacrosomal space. Certain mitochondria move posteriorly towards the nucleus, thus constituting the spermatozoon midpiece. The flagellum, originated from the distal centriole, appears in the early spermiogenesis. Ultimately, mature spermatidium dissociates into numerous spermatozoa, which are subsequently released as a single cell from the coelom into the nephridia. The spermatozoon has a prominent head, containing an acrosome and nucleus, a short midpiece and a slender tail. When compared with other sipunculans or invertebrates with external fertilization, the spermiogenesis of P. esculenta, presumably, is closely associated with its biological adaptations for the reproductive strategy.

Etoposide interferes with the process of chromatin condensation during alga Chara vulgaris spermiogenesis

DNA topoisomerase II plays an essential role in animal spermiogenesis, where changes of chromatin structure are connected with appearance of transient DNA breaks. Such topo II activity can be curtailed by inhibitors such as etoposide and suramine. The aim of the present study was to investigate, for the first time, the effect of etoposide on spermatid chromatin remodeling in the green alga Chara vulgaris. This inhibitor prolonged the early spermiogenesis stages and blocked the formation of the phosphorylated form of histone H2AX at stages VI–VII. The lack of transient DSBs at these stages impairs the elimination of supercoils containing nucleosomes which lead to disturbances in nucleoprotein exchange and the pattern of spermatid chromatin fibrils at stages VI–VIII. Immunofluorescent and ultrastructural observations revealed that during C. vulgaris spermiogenesis topo II played an important role similar to that in mammals. Some corresponding features had been pointed out before, the present studies showed further similarities.

Ultrastructural, autoradiographic and electrophoretic examinations of Chara tomentosa spermiogenesis

Ultrastructure of a spermatid nucleus changes many times during spermiogenesis. Condensed chromatin forms irregular clusters during phases I-II, a continuous ring adjacent to a nuclear envelope during phases III-V and a network occupying the whole nucleus during phase VI. In advanced spermiogenesis dense chromatin disappears and short randomly positioned fibrils arise, then long parallel ones are found (phase VIII) which during phase IX form a lamellar structure. In mature spermatozoids (phase X) chromatin becomes extremely condensed. <sup>3</sup>H-arginine and <sup>3</sup>H-lysine incorporation into spermatids during 2-min incubation is intensive during phases IN, decreases during phases VI, VII and becomes very low during phases VIII-IX. Capillary electrophoresis has shown that during <em>Chara tomentosa</em> spermiogenesis replacement of histones with basic proteins whose mobility is comparable to that of salmon protamines takes place. At the beginning of spermiogenesis core and linker histones are found in spermatids. During early spermiogenesis protamine-like proteins appear and their amount increases in late spermiogenesis when core histones are still present. In mature spermatozoids only protamine-like proteins represented by 3 fractions: 9.1 kDa, 9.6 kDa, 11.2 kDa are found. Disappearance of linker histones following their modification precedes disappearance of core histones. The results indicate that dynamic rearrangement of chromatin ultrastructure and aminoacid incorporation rate during spermiogenesis are reflected in basic nuclear protein changes.

Bioinformatic identification of novel elements potentially involved in messenger RNA fate control during spermatogenesis.

In eukaryotic cells, 3' untranslated regions (3' UTRs) of mRNA transcripts contain conserved sequence elements (motifs), which, once bound by RNA-binding proteins, can affect mRNA stability and translational efficacy. Despite abundant sequences contained within the 3' UTRs, only a limited number of motifs are known to interact with RNA-binding proteins and have a role in mRNA fate control. Spermatogenesis represents an excellent in vivo model for studying posttranscriptional regulation of gene expression because numerous mRNAs are transcribed in late pachytene spermatocytes and/or round spermatids, but their translation will not occur until many hours or even days later, when they have developed into elongated spermatids, in which transcription has long been shut off because of the increasingly condensed chromatin. Translationally suppressed mRNAs are sequestered and confined to ribonuclear protein particles, and their loading onto the ribosomes marks their translation. By bioinformatic sequence analyses of the 3' UTRs of translationally suppressed mRNAs during spermatogenesis, we identified numerous novel sequence elements overrepresented in the transcripts subject to posttranscriptional regulation than in the unregulated transcripts. These include AU(U/A)(U/A)UGAGU and (A/U)AUUA(U/C/G) for genes translationally upregulated in early spermiogenesis, and (G/A)GUACG(U/C/A)(A/U)(A/U) and UGUAGC for genes translationally upregulated in late spermiogenesis. The bioinformatic approach reported in this study can be adapted for rapid discovery of novel regulatory elements involved in mRNA fate control in a wide range of tissues or organs.

Ultrastructural analysis of the nucleolar aspects at spermiogenesis in triatomines (Heteroptera, Triatominae)

In this study the ultrastructural technique was used to analyze seminiferous tubule cells of the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans. The data obtained provided evidence of the phenomenon known as persistence of the nucleolar material in initial spermatids at early differentiation. Our results confirmed the presence of the nucleolus and its products during spermiogenesis up to the formation of the axoneme and during spermatid elongation in all three species studied, similar to the process that takes place during cell division. In early spermatids, the nucleoli had a reticulate appearance and a well defined nucleolonema in P. megistus; showed a clear distinction between the fibrillar and the granular component in T. infestans; and had a compact aspect in R. pallescens. In this study, ultrastructural analyses at spermiogenesis indicated that these nucleolar products may represent RNP complexes that will probably be needed at early spermiogenesis when important changes such as chromatin condensation and acrosome and flagellum formation take place. Therefore, it was concluded from the ultrastructural analysis that the triatomine nucleolus does not totally disappear but remains as corpuscles that gather to form the next nucleolar cycle that in the case of meiosis, will be completed if fertilization occurs and a zygote is formed.

312 MORPHOLOGICAL AND MORPHOMETRICS CHARACTERISTICS OF NUCLEAR SPERM IN THE EPIDIDYMAL TRANSIT OF BULLS

During the transit through the epididymis, many morphological, physiological, and biochemical characteristics of spermatozoa are modified, as part of the maturation process. Nuclear maturation continues in the epididymis through an increase in formation of protamine disulfide. Thus, penetration through the oocyte membranes could be facilitated for elongated spermatozoa with dramatically condensed chromatin and nuclear integrity. Therefore, size, shape, and nuclear defects could be used to estimate the stage-related nuclear transformations from early spermiogenesis to the end of epididymal transit. Nellore bulls (n = 9), 30-36 months old, with high seminal quality (>80% motile and morphologically normal sperm) were submitted to orchiectomy. Impressions in slides of the caput, corpus, and caudal regions of the epididymis were prepared for evaluation of morphology (Feulgen stain, phase-contrast microscopy at 1000 ×) and morphometry of the nuclear sperm. The slides were captured in a Motic 2300 camera adapted to the microscope and digitally assessed. Nuclear morphology was considered normal (without visible alterations), head defect (variations in shape and form), and nuclear defects (abnormal chromatin condensation and presence of vacuoles). Base, width, length (μm), and area (μm2) were estimated in least 60 sperm nuclei. No difference (P > 0.05) among epididymal regions for normal nuclei (70.3 ± 3.1%), head defects (3.4 ± 0.5%), and nuclear defects (5.3 ± 1.3%) were seen. The base was higher (2.68 ± 0.5 μm, P < 0.01) in the caput than corpus (2.44 ± 0.4 μm) and caudal regions (2.41 ± 0.4μm). Normal nuclei were associated (P < 0.01) with width (r = 0.20), length (r = 0.27), and area (r = 0.44) in the caput and with width (r = 0.21), length (r = 0.40), and area (r = 0.33) in the corpus of epididymis. Epididymal transit affected (P < 0.001) the measures and nuclear status that accounted for regression analysis: normal nucleus (40.254 + 3.027, length; R2 = 0.20), head defects (0.922 + 1.097, width + 0.093 × area; R2 = 0.26), and nuclear defects (6.993-0.496, length + 0.454, base; R2 = 0.23). The results suggest that important variations occur in the nuclear status during the epididymal transit in the bovine spermatozoa. The higher measures in the sperm base of the caput suggest a narrowing probably indicating the continuity of nuclear remodeling from the final steps of spermiogenesis. The area accounts for 20% of the nuclear shape variations along the epididymal segment. These events characterize the adaptation of nuclear membranes and chromatin structure surrounded by epididymal environment and comprise a part of the maturation process. Additionally, morphometric variations are associated with defects in the nuclear structures and can be used to determine the conditions of spermatogenesis and sperm maturation based on the evaluation of ejaculated semen. We thank CNPq/PROPP and Fundect for financial support.

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 2: Changes in spermatid organelles associated with development of spermatozoa

Spermiogenesis is a long process whereby haploid spermatids derived from the meiotic divisions of spermatocytes undergo metamorphosis into spermatozoa. It is subdivided into distinct steps with 19 being identified in rats, 16 in mouse and 8 in humans. Spermiogenesis extends over 22.7 days in rats and 21.6 days in humans. In this part, we review several key events that take place during the development of spermatids from a structural and functional point of view. During early spermiogenesis, the Golgi apparatus forms the acrosome, a lysosome-like membrane bound organelle involved in fertilization. The endoplasmic reticulum undergoes several topographical and structural modifications including the formation of the radial body and annulate lamellae. The chromatoid body is fully developed and undergoes structural and functional modifications at this time. It is suspected to be involved in RNA storing and processing. The shape of the spermatid head undergoes extensive structural changes that are species-specific, and the nuclear chromatin becomes compacted to accommodate the stream-lined appearance of the sperm head. Microtubules become organized to form a curtain or manchette that associates with spermatids at specific steps of their development. It is involved in maintenance of the sperm head shape and trafficking of proteins in the spermatid cytoplasm. During spermiogenesis, many genes/proteins have been implicated in the diverse dynamic events occurring at this time of development of germ cells and the absence of some of these have been shown to result in subfertility or infertility.

Spermiogenic nuclear protein transitions and chromatin condensation. Proposal for an ancestral model of nuclear spermiogenesis

We have chosen three species (Sparus aurata, Dicentrarchus labrax, and Monodonta turbinata) that represent different transition patterns in the composition and structure of spermiogenic nuclei. The transition patterns of these species are representative of spermiogenesis in a large number of animal species. We analyze: (a) nuclear protein exchange; (b) chromatin condensation pattern; and (c) histone acetylation during spermiogenic development. In the simplest spermiogenesis histones and nucleosomes remain in mature sperm. Chromatin of spermatids is organized into 20 nm granules, simultaneous with a nuclear volume reduction. The granules coalesce in the final stage of spermiogenesis. Granular chromatin is correlated with acetylation of histones H3 and H4, whereas final coalescence is associated with histone deacetylation. We also studied two other spermiogenesis where a basic protein substitutes histones. Each species has a very different substituting protein. One has a typical protamine of 34 amino acids; the other has a sperm nuclear basic proteins (SNBP) of 106 amino acids. In both, the structural transitions and histone acetylation pattern are similar: in early spermiogenesis chromatin is organized into 20 nm granules, and histones are significantly acetylated, while the nuclear volume decreases. Subsequently, acetylated histones are displaced by the protamine or SNBP. Histone substitution causes chromatin remodelling and additional reduction in nuclear volume. We analyze these three cases together with earlier works and propose that the formation of 20 nm granules containing acetylated H3 and H4 accomplishes the minimum functional requirement to be considered the most evolutionarily ancestral chromatin conformation preceding condensation in animal spermiogenesis.